Mitochondria‐Targeted Nanoadjuvants Induced Multi‐Functional Immune‐Microenvironment Remodeling to Sensitize Tumor Radio‐Immunotherapy

Abstract It is newly revealed that collagen works as a physical barrier to tumor immune infiltration, oxygen perfusion, and immune depressor in solid tumors. Meanwhile, after radiotherapy (RT), the programmed death ligand‐1 (PD‐L1) overexpression and transforming growth factor‐β (TGF‐β) excessive secretion would accelerate DNA damage repair and trigger T cell exclusion to limit RT efficacy. However, existing drugs or nanoparticles can hardly address these obstacles of highly effective RT simultaneously, effectively, and easily. In this study, it is revealed that inducing mitochondria dysfunction by using oxidative phosphorylation inhibitors like Lonidamine (LND) can serve as a highly effective multi‐immune pathway regulation strategy through PD‐L1, collagen, and TGF‐β co‐depression. Then, IR‐LND is prepared by combining the mitochondria‐targeted molecule IR‐68 with LND, which then is loaded with liposomes (Lip) to create IR‐LND@Lip nanoadjuvants. By doing this, IR‐LND@Lip more effectively sensitizes RT by generating more DNA damage and transforming cold tumors into hot ones through immune activation by PD‐L1, collagen, and TGF‐β co‐inhibition. In conclusion, the combined treatment of RT and IR‐LND@Lip ultimately almost completely suppressed the growth of bladder tumors and breast tumors.


Figure S1 .
Figure S1.(A) Detection of the expression of COL1A1, α-SMA, and PD-L1 protein in MB49 cells by western blotting after treatment with indicated doses of LND (n = 3).(B-D) The quantitative analysis of the expression of COL1A1, α-SMA, and PD-L1 protein was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05.

Figure S6 .
Figure S6.The standard curve of IR-LND was examined by UV-vis spectrophotometry.

Figure S8 .
Figure S8.(A) Detection of the expression of HIF-1α and PD-L1 protein in MB49 cells by western blotting after treatment with indicated doses of IR-LND@Lip (n = 3).(B-C) The quantitative analysis of the expression of HIF-1α or PD-L1 protein was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01, *** p < 0.001.

Figure
Figure S9.(A) Detection of the expression of p-AMPK and AMPK protein in 4T1 cells by western blotting after treatment with indicated doses of IR-LND@Lip (n = 3).(B) The quantitative analysis of the expression of p-AMPK/AMPK was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.*** p < 0.001.

Figure
Figure S11.(A) Detection of the expression of PD-L1 protein in 4T1 or MB49 cells by western blotting after treatment with different radiation doses (n = 3).(B-C) The quantitative analysis of the expression of PD-L1 proteins in 4T1 or MB49 cells was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01.

Figure S12 .
Figure S12.The quantitative analysis of the expression of PD-L1 proteins in 4T1 cells after different treatments was performed by ImageJ (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05, ** p < 0.01, NS indicates no statistically significant difference (p > 0.05).

Figure S13 .
Figure S13.(A) Detection of the expression of PD-L1 protein in MB49 cells by western blotting after different treatments (n = 3).(B) The quantitative analysis of the expression of PD-L1 proteins in MB49 cells after different treatments was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05, ** p < 0.01.

Figure S14 .
Figure S14.The quantitative analysis of the 4T1 cell clones after different treatments with IR-LND@Lip (1 μM) with or without RT co-treatment (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05, ** p < 0.01.

Figure S15 .
Figure S15.The quantitative analysis of IR-LND@Lip and RT on T cell killing of tumor cells (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.*** p < 0.001.

Figure S16 .
Figure S16.The quantitative analysis of the expression of TGF-β protein in 4T1 cells after treatment with indicated doses of IR-LND was performed by ImageJ (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01.

Figure S17 .
Figure S17.(A) Detection of the expression of TGF-β protein in MB49 cells by western blotting after treatment with indicated doses of IR-LND (n = 3).(B) The quantitative analysis of the expression of TGF-β protein in MB49 cells after treatment with indicated doses of IR-LND was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01, *** p < 0.001.

Figure S18 .
Figure S18.The quantitative analysis of the expression of TGF-β protein in 4T1 cells after different treatments was performed by ImageJ (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01.

Figure S19 .
Figure S19.(A) Detection of the expression of TGF-β protein in MB49 cells by western blotting after different treatments (n = 3).(B) The quantitative analysis of the expression of TGF-β protein in MB49 cells after different treatments was performed by ImageJ.Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01.

Figure S20 .
Figure S20.(A-B) Detection of the expression of TGF-β protein in MB49 cells by western blotting after treatment with different radiation doses and further quantification by Image J (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.** p < 0.01, *** p < 0.001.

Figure S21 .
Figure S21.(A-B) Detection of the expression of TGF-β protein in MB49 cells by western blotting after different treatments with indicated doses of IR-LND@Lip (0 or 1 μM) with or without RT co-treatment and further quantification by Image J (n = 3).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05, ** p < 0.01.

Figure S22 .
Figure S22.(A-C) Detection of the expression of COL1A1 and α-SMA protein in fibroblasts by western blotting after indicated treatments and further quantification by Image J (n = 3).CM stands for the conditional culture medium (a 1:1 mixture of complete culture medium with 20% FBS and culture medium derived from irradiated 4T1 cells).Data were demonstrated as mean ± SD.Statistical analysis was performed via the two-tailed Student's t-test.* p < 0.05, ** p < 0.01.

Figure S23 .
Figure S23.Representative H&E staining images in 4T1 tumors collected from the mice after different treatments, scale bar = 100 μm.

Figure S25 .
Figure S25.Primary tumor growth curves of each mouse in different treatment groups within 14 days.